Proceedings Volume 2010 (format .pdf) - SimpBTH

The aim of this paper is to use the original direct viable counts method (Kogure etal., 1979) to count cells that are capable of division in the presence of gasoline, andto couple this method with automated image analysis.MATERIALS AND METHODSSamples and microcosms were as follows: we collected water samples in volume300 mL of natural sea water from the Black Sea (0.5m depth; Tomis seaport,44 o 10 ’ 44 ’’ N; 28 o 39 ’ 32 ’’ E) in sterile Polyethylene transparent bottles, naturalsample- control (M3); control supplemented with petroleum hydrocarbons(gasoline-0.5% v/w) (M2) and iii) control supplemented with petroleumhydrocarbons (gasoline-0.5% v/w) and nutrients (ammonium nitrate 0.005% w/w)(M1). Variables where with respect to: i) nature and concentration of nutrients; ii)temperature of incubation (18ºC- fluorescent light); iii) the use of filtered(Millipore membranes 0.45 µm) seawater, in order to eliminate bacteriovorusmicroorganisms (Ghita & Ardelean, 2010). At different interval of time, sampleswere collected in sterile Falcon tubes, fixed with buffered formaline (2% finalconcentration) and kept at 4 o C in darkness without any added fluorochrome. Thisfixation has been made to be measured other parameters (Ardelean et al., 2009b).In parallel, for preliminary experiments, one have collected in the same locationsmentioned above fresh seawater sample (S1), which was supplemented with yeastextract to 50 mg / L final concentration (S2), or with yeast extract 50 mg / L andnalidixic acid (20mg/L), and incubated 6 hours at 30°C in the dark .Direct viable counts. The method of direct enumeration of viable cells is based onincubation of natural samples in the presence of an antibiotic (nalidixic acid asspecific inhibitor of DNA synthesis, without affecting other metabolic processes)and nutrients (yeast extract). Active cells may continue to metabolize nutrients andthus become more elongated after incubation period. Sterile flasks containingseawater samples (40mL) were kept at a constant temperature (30°C) incubated indark and continuous stirring. Samples from each microcosms were supplementedwith yeast extract (50 mg / L final concentration), nalidixic acid (20 mg / L finalconcentration) (Kogure et al., 1979) and gasoline (0.5% final concentration).Subsequently samples were harvested each two hours (considering the time T o , T 1–after 2 hours, T 2 - after 4 hours; T 3 - after 6 hours, T 4 - after 8 hours). Then sampleswere stained with acridine orange and visualized by epifluorescence microscopy.Total cell counting and DVC were done tacking into account 15 fields per sampleand 120 fields per sample, respectively.Acridine Orange and Propidiu Iodide direct counts. Aliquots with volume of 5ml were stained with 10 µl Acridine Orange (0.1mg/mL final concentration) andfiltered on Isopore polycarbonate membrane filters (0.2 µm pore size, black) andwashed with 10 ml distilled water (Robertson & Button, 1989; Jansson & Prosser,1997; Kawai et al. 1999; Marie et al., 1999). Staining time was 5 minutes. PI205